Ethanol and higher n-chain alcohols stimulate homomeric 5- Hyrdroxytryptamine3A receptor (5-HT3A) function. Work on this receptor, as well as studies in other laboratories on related ligand-gated ion channels, suggests that the second transmembrane domains (TM2) of these receptors play a critical role in alcohol action. However, it remains unknown whether identified residues in TMZ are part of a critical binding domain for alcohols or whether they play a key role in the transduction of n-chain alcohol action. A third possibility is that a mutation in TM2 could obscure the effects of alcohols through a kinetic mechanism. The major goal of this proposal is to delineate which of these three mechanisms is responsible for altering the alcohol sensitivity of 5-HT3A receptors mutated in the TM2 domain. The alcohol sensitivity of wild-type and mutant 5-HT3A receptors will be assessed in oocytes with the two-electrode voltage clamp technique and in HEK293 cells with patch-clamp technology and standard perfusion and ultra-fast superfusion drug application methods. Residues lining the channel pore will be identified and eliminated from study through alanine scanning mutagenesis and cysteine substitution methods. Changes in secondary structure will be assessed with tryptophan scanning mutagenesis and discrete Fourier transformation analysis. Residues facing other transmembrane domains will be mutated to amino acids differing in physicochemical properties and will be examined for differences in alcohol action. False positive receptors, whose normal gating properties are changed by mutagenesis, will be identified and eliminated if they have altered responsiveness to battery of 5-HT3A receptor drugs acting at different loci within the receptor. Candidate alcohol binding domains can be preliminarily separated from alcohol transduction sites through correlation of changes in alcohol sensitivity with physicochemical properties of substituted amino acids, changes in channel kinetics, and alteration in alcohol cut-off. Wild-type/mutant titration experiments will be used to determine the minimum number of mutant subunits required per receptor to achieve the mutant alcohol phenotype; a dominant mutant phenotype would suggest that an alcohol transduction site has been identified.